Method for enriching optical isomers by means of simulated mobile bed

ABSTRACT

The invention concerns a method for separating from a mixture two optical isomers using a simulated moving bed including an assembly of columns divided into at least three successive zones. The amount of charge injected is greater than that of the state of a predetermined reference state. The discharge flow is greater than that of the predetermined reference state if it is required at least to preserve the purity of the less immobilized enantiomer. The discharge flow is less than that of the reference state if it is required to preserve the purity of the more immobilized enantiomer.

REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/FR98/00550 filed Mar. 19, 1998.

The present invention relates to a method intended for the separation ofa mixture of two optical isomers, by means of a simulated mobile bed(SMB) using a chiral stationary phase and an achiral eluent.

It is known that, in chiral chromatography, porous solids are used withwhich a mixture of two optical isomers dissolved in a fluid, or amixture of fluids, is placed in contact, which present differentproperties of adsorption having regard to each of the optical isomers.These porous solids are called chiral stationary phases or CSPs.

Most of the time, chiral chromatography methods employ a simple column,filled with chiral stationary phase, through which a fluid, calledeluent, passes. At regular intervals of time, there is injected into thestream of eluent the mixture of the optical isomers to be separated,which is dissolved in an appropriate fluid, which may in particular beconstituted by the eluent itself The mixture of optical isomers is thusentrained in the column and, at the outlet thereof, the optical isomerleast adsorbed is, in known manner, recovered before the most adsorbedoptical isomer.

In order to ensure separation of two optical isomers, it has also beenproposed to employ a so-called “simulated mobile bed” technique. Thesimulated mobile bed is an old concept which has been used since the1960s for the large-scale separation of the isomers of xylene,n-alkanes, iso-alkanes or fructose and glucose. Since 1990, it has beenused in the domain of fine chemistry, and especially for the separationof optical isomers. Concerning this latter category of products, thesimulated mobile bed is described as being an effective method forsimultaneously obtaining two very pure optical isomers. Purities thatmay exceed 99.5% have thus been obtained. This technique has beendescribed in particular in U.S. Pat. Nos. 2,985,589, 4,402,832,4,498,991 and 5,126,055.

It is known that a simulated mobile bed is constituted by a given numberof columns containing a stationary phase which are connected together inseries. A solution, constituted by the mixture of the compounds to beseparated dissolved in an appropriate fluid, called charge, and aneluent, are injected continuously at the inlet of two different columns.At the level of a column located downstream of the column where thecharge is injected, a flux, called raffinate, is collected, whichcontains the enantiomer less immobilised in the eluent and, upstream ofthe column where the charge is injected, a flux, called discharge, iscollected, which contains the enantiomer more immobilised in the eluent.In this way, a plurality of work zones are defined, each comprising apoint of injection and a point of drawing-off.

The points of injection and of drawing-off are offset at regularintervals of time in the direction of flow. The interval of time locatedbetween two offsets of the points of injection/drawing-off is called aperiod.

Principally, two configurations of simulated mobile bed have beendescribed: a simulated mobile bed with four zones and a simulated mobilebed with three zones (cf. Ruthven and Ching 1989 “Chemical EngineeringScience”).

A major drawback of this type of technique is that, beyond a givenproductivity, called maximum productivity of the installation, anyincrease in the amount of charge is made to the detriment of the purityof the products obtained, this limiting the development of thetechniques of this type.

Applicants have established that it was possible to exceed the maximumproductivity obtained in the simulated mobile beds functioning inaccordance with the methods of the prior state of the art, whileconserving the purity of one or the other of the separated enantiomers.In order to achieve such a result, Applicants have established thatappropriate modifications of the functions exerted at the level of thedifferent zones of the simulated mobile bed as well as of the flowratesemployed were necessary.

The tests carried out by Applicants have thus led them to establishthat, in order to improve the productivity of the installation, theamount of charge had, of course, to be increased but that, in order toobtain results likewise interesting from the standpoint of purity of theseparated enantiomers, such increase had to be combined with anappropriate modification of the discharge flow.

The present invention thus has for its object to propose a method ofseparation of optical isomers which makes it possible to improve theyield of the methods according to the prior state of the art, whileconserving the purity of one of the isomers obtained, and even byincreasing the latter.

The present invention thus relates to a method of separation of amixture of two optical isomers adapted to be more or less immobilised ina chiral stationary phase, this method being of the type employing asimulated mobile bed, constituted by an assembly of columns disposed inseries, filled with a chiral stationary phase, which are divided into atleast three successive zones, namely a first zone at the inlet of whichan eluent is injected and at the outlet of which a flux, calleddischarge flux, principally containing the more immobilised enantiomer,is drawn off, a second zone at the inlet of which said mixture isinjected, and a third zone at the outlet of which a flux, calledraffinate flux, principally containing the less immobilized enantiomeris drawn off, characterized in that, with respect to a reference statein which: a) the optical isomer more immobilised in the stationary phaseis completely desorbed in the first zone and is completely adsorbed inthe third zone and where the optical isomer less immobilised in thestationary phase is completely desorbed in the second zone, and b) theamount of charge is maximum,

the amount of charge injected is greater than that of the referencestate,

the discharge flow is greater than that of the reference state if it isdesired at least to preserve the purity of the less immobilisedenantiomer, or the discharge flow is less than that of the referencestate if it is desired at least to preserve the purity of the moreimmobilised enantiomer.

In one form of embodiment of the invention, the amount of chargeinjected is rendered greater than that of the reference state by playingon the volumic flowrate of the charge.

In another form of embodiment of the invention, the amount of chargeinjected is rendered greater than that of the reference state by playingon the concentration of the charge.

Preferably, and as set forth in the form of embodiment describedhereinafter, the simulated mobile bed comprises four zones, the fourthzone being located downstream of the point of drawing off of raffinateand, in the reference state, the optical isomer less immobilised in thestationary phase is completely adsorbed in this fourth zone. The outletof the last zone is possibly connected to the inlet of the first zone.

Furthermore, it has been established that, at first approximation, thefactor of increase of amount of charge, i.e. the ratio of the amount ofcharge injected according to the invention with respect to the amount ofcharge injected in the reference state, for narrow intervals of thisfactor, and the factor of correction of discharge flow, i.e. the ratioof the discharge flow with respect to the ratio of the discharge flow inthe reference state, are bound by a substantially linear mathematicalrelationship. It will be noted that, under these conditions, the slopeof the curve representing the variation of the factor of correction ofdischarge flow as a function of the variations of the factor of increaseof amount of charge varies inversely with respect to the value of theselectivity of the stationary phase used.

More precisely, it has been established that the factor of increase ofthe amount of charge and the factor of correction of discharge flow arebound by a linear relationship expressing the ratio of the factor ofincrease of the amount of charge with respect to the factor ofcorrection of discharge flow decreased by a value of one, as a functionof the factor of correction of charge flow decreased by a value of one.It has also been established that the factor of proportionality of saidlinear relationship includes the value of the selectivity of thestationary phase used. More precisely, said factor of proportionality issubstantially equal to a quarter of the value of the selectivity.

The present invention is applicable to a simulated mobile bed systemconstituted by a given number of columns disposed in series in zonesallocated different functionalities. The mixture of the optical isomersthat it is desired to separate and one or two achiral desorbents arecontinuously introduced in the columns containing an optically activestationary phase. Two streams containing the enriched optical isomersare extracted continuously from the line of columns. The charge and thedesorbent or desorbents are injected via points of injection which areoffset periodically in the direction of displacement of the eluent. Thetwo flows of enriched optical isomers are recovered in periodicallyoffset points of recovery.

A form of embodiment of the present invention will be describedhereinafter by way of example, with reference to the accompanyingdrawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a series of columns constituting asimulated mobile bed enabling the method according to the invention tobe carried out.

FIGS. 2 to 5 are graphs which show the concentration of more immobilisedenantiomer (curves a) and of less immobilised enantiomers (curves b) atthe inlet of each of the columns which constitute the simulated mobilebed, on the one hand in the case of a method according to the priorstate of the art (curves in broken lines), in the case of the maximumpossible productivity with this type of technique being attained, and,on the other hand, according to the invention (curves in solid lines),in the case of the productivity of the less immobilised enantiomer beingincreased.

FIG. 6 is a graph representing the variation of the factor of correctionof discharge flow as a function of the factor of increase of the amountof charge, for various values of the selectivity of the stationaryphase.

FIG. 7 is a graph representing the variation of the ratio of the factorof increase of the amount of charge with respect to the factor ofcorrection of discharge flow decreased by a value of one, as a functionof the factor of increase of the amount of charge decreased by a valueof one, for five values of the selectivity of the stationary phase.

FIG. 8 is a graph representing the variation of the slope of thefunction represented in FIG. 7 as a function of the selectivity of thestationary phase.

DESCRIPTION OF PREFERRED EMBODIMENT

FIG. 1 shows by way of example an installation intended to ensure theseparation of two optical isomers by means of a simulated mobile beddevice, which is constituted in the present case by a series of eightcolumns C₁, C₂, . . . , C₈ which are divided into four zones of twocolumns I, II, III, IV, these numberings being made in the direction offlow.

Zone I extends over the first two columns C₁ and C₂ between a point 3,where the eluent is injected via a conduit 1 and a point 5, where themost adsorbed optical isomer is drawn off, in the discharge flow via aconduit 7.

Zone II extends over columns C₃ and C₄ namely between the drawing offpoint 5 and a point 9 where the charge is continuously injected via aconduit 11, which charge is constituted by the mixture of the opticalisomers which it is desired to separate which are dissolved in adiluent, which may in particular be constituted by the eluent injectedat point 3.

Zone III extends over columns C5 and C6, namely between the point ofinjection 9 of the charge and a point 13 where the least adsorbedoptical isomer is drawn off, in the flow of the raffinate via a conduit15.

Zone IV extends over columns C7 and C8, namely between the drawing offpoint 13 of the raffinate and the point of injection 3 of the eluent.The outlet of column C8 is connected to the inlet of column C1.

In a functioning of conventional type, in simulated mobile bed for achiral separation, flows Q_(I), Q_(II), Q_(III) and Q_(IV) in each ofthe respective zones I to IV are chosen in the following manner:

flow Q_(I) is such that the more immobilised optical isomer iscompletely desorbed in zone I,

flow Q_(II) is such that the less immobilised optical isomer iscompletely desorbed in zone II,

flow Q_(III) is such that the more immobilised optical isomer iscompletely adsorbed in zone III,

flow Q_(IV) is such that the less immobilised optical isomer iscompletely adsorbed in zone IV.

The preceding conditions lead to several possible operational states.From these different possible states, the person skilled in the art candetermine the one which leads to the highest productivity. This statewill hereinafter be called “reference state”.

Four examples of embodiment of the invention will be describedhereinafter:

EXAMPLE I

In a first example of embodiment of the invention, it is proposed toseparate by means of such a simulated mobile bed, the enantiomers of(+/−)-5-[1,2,3,4-tetrahydro-6-chinolyl]-6-methyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-onwhich is dissolved in a solvent constituted by methanol at aconcentration of 6 g/l. It was desired, during this operation, tocollect the less immobilised enantiomer. To that end, a simulated mobilebed installation of the type described hereinabove was used, comprisingeight columns divided into four work zones I, II, III, IV, as mentionedpreviously.

The stationary phase was a chiral phase of the tri (p-methyl) benzoateof cellulose type whose granulometry was from 20 to 45 micrometers. Thediameter of each column was 26 mm and its length 10 cm. The eluentinjected at point 3 was methanol. The temperature of the treatment was25° C.

The starting point, or reference state, was determined by the conditionsaccording to the prior state of the art in which a maximum ofproductivity is obtained. In this reference state, the flow of thecharge injected at point 9 was 6.2 ml/min, the drawn off discharge flowat point 5 was 24.1 ml/min, the flow of drawn off raffinate at point 13was 6.1 ml/min, the flow of eluent injected at point 3 was 24 ml/min,the flow Q₁ was 44.9 ml/min and the period was 14.6 min.

The purity of the less immobilised enantiomer which was drawn off atpoint 13, in the raffinate, was 99.2% (3 g/l) and the purity of the moreimmobilised enantiomer which was drawn off at point 5, from thedischarge, was 98.2%. The productivity was 18.5 mg/min of lessimmobilised enantiomer.

According to the invention, the amount of charge injected at point 9 wasincreased to take it to a value of 9.3 ml/min, viz. a factor of increaseof the amount of charge γ of 1.5, which was obtained by increasing theflow of the charge, and, correlatively, the discharge flow was increasedby a factor of 1.2, hereinafter called factor of correction of dischargeflow β. The purity and productivity of the enantiomer desired in thepresent case was measured, namely the less immobilised enantiomer. Itwas thus ascertained that 23.2 mg/min of this enantiomer with a purityof 99.5% was obtained.

FIG. 2 shows the curves of variation of the concentration C of moreimmobilised enantiomer (curve a) and of less immobilised enantiomer(curve b) in simulated mobile bed devices respectively according to theprior state of the art (curves in broken lines) and according to theinvention (curves in solid lines), at the level of the inlet of each ofthe eight columns C1 to C8 of the device.

It is thus observed in FIG. 2 that, at the inlet of column C7, theconcentration of more immobilised enantiomer is virtually zero, thusconfirming the great purity obtained for the less immobilisedenantiomer. It will also be noted that at the inlet of column C3, i.e.at the point of drawing off of the extract (the more immobilisedenantiomer), the composition of less immobilised enantiomer according tothe invention (curves in solid lines) is not zero as in the prior stateof the art (curves in broken lines), which explains the reduced purityof the more immobilised enantiomer.

EXAMPLE II

A second example of embodiment of the invention will be describedhereinafter, in which it is proposed to separate a racemic, namelybinaphtol, which is dissolved in a solvent, which the the eluent used inthe method, and which is constituted by an equivolume mixture of heptaneand of isopropanol, the concentration of the charge thus constitutedbeing 8 gl.

During this operation, it is desired to collect the less immobilisedenantiomer. The method was carried out in a simulated mobile bedidentical to that described previously.

Conditions of Implementation

Racemic Binaphtol Selectivity 1.45 Diluent Equivolume mixture of heptaneand of isopropanol Concentration of the racemic 8 g/l Nature of theeluent Equivolume mixture of heptane and of isopropanol Temperature ofthe treatment 20° C. Stationary phase tri(3,5 dimethylphenyl carbamate)of amylose marketed under the Trademark “CHIRALPAK AD” Sought enantiomerThe less immobilised one

Reference State

Flow of the charge injected at point 9  3.4 ml/min Discharge flow drawnoff at point 5 19.7 ml/min Flow of raffinate drawn off at point 13  5.4ml/min Flow of eluent injected at point 3 21.7 ml/min Flow in zone 1  70 ml/min Period  1.6 min Composition of the flow of raffinate 98.6%of the less drawn off at point 13 immobilised enantiomer Productivity ofthe less immobilised 13.3 mg/min enantiomer

According to the invention, the amount of charge introduced at point 9was multiplied by a factor of increase in amount of charge y equal to 3and the amount of extract drawn off at point 5 was also increased.

The other operational conditions, namely the temperature of treatment,the flow of the raffinate drawn off at point 13, were preservedunchanged.

It has been observed that, under the conditions of the invention foroperating the simulated mobile bed with an optimum yield, as the personskilled in the art knows how to do, the amount of extract injected hadin that case to be multiplied by a factor of correction of dischargeflow β, equal to 1.75.

Under these conditions, a productivity of the less immobilisedenantiomer of 25 mg/min with a purity of 99.5% was obtained.

FIG. 3 shows the curves of variation in the concentration C of moreimmobilised enantiomer (curve a) and of less immobilised enantiomer(curve b) in simulated mobile bed devices respectively according to theprior state of the art (curves in broken lines) and according to theinvention (curves in solid lines), at the level of the inlet of each ofthe eight columns C1 to C8 of the device.

It is thus ascertained that the present invention makes it possible toimprove not only the productivity of one of the two enantiomers asdesired, but also the purity thereof.

EXAMPLE III

A third example of embodiment of the invention will be describedhereinafter in which it is proposed to effect the separation of aracemic ester.

Conditions of Implementation

Racemic Racemic ester Selectivity 1.3 Diluent n-heptane/isopropanolConcentration of the racemic 3 g/l Nature of the eluentn-heptane/isopropanol Temperature of the treatment 25° C. Stationaryphase tri(p-methyl benzoate) of cellulose Sought enantiomer The lessimmobilised one

Reference state

Flow of the charge injected at point 9  4.3 ml/min Discharge flow drawnoff at point 5 15.1 ml/min Flow of raffinate drawn off at point 13  5.6ml/min Flow of eluent injected at point 3 16.4 ml/min Flow in zone 1  50 ml/min Period  9.6 min Composition of the flow of raffinate 98.7%of the less drawn off at point 13 immobilised enantiomer Productivity ofthe less immobilised  6.3 mg/min enantiomer

According to the invention and, as previously, the amount of chargeintroduced at point 9 was multiplied by a factor of increase of amountof charge γ, equal to 1.3, and the amount of extract drawn off at point5 was also increased.

The other operational conditions, namely the temperature of treatment,the flow of the raffinate drawn off at point 13, were preservedunchanged.

It was ascertained that, under the conditions of the invention foroperating the simulated mobile bed with an optimum yield, as the personskilled in the art knows how to do, the amount of extract injected hadin that case to be multiplied by a factor of correction of dischargeflow β, equal to 1.20.

Under these conditions, a productivity of the less immobilisedenantiomer of 8 mg/min with a purity of 98.9% was obtained, the purityin the extract of the more immobilised enantiomer being 95.1%.

FIG. 4 shows the curves of variation in the concentration C of moreimmobilised enantiomer (curve a) and of less immobilised enantiomer(curve b) in simulated mobile bed devices respectively according to theprior state of the art (curves in broken lines) and according to theinvention (curves in solid lines), at the level of the inlet of each ofthe eight columns C1 to C8 of the device.

EXAMPLE IV

In a fourth example of embodiment of the invention, the amount of chargeintroduced at point 9 was multiplied, from the same enantiomer and thesame reference state as that of Example III, by a factor of increase ofthe amount of charge γ equal to 2, all the other operational parametersof the method remaining unchanged.

It was ascertained that, under the conditions of the invention foroperating the simulated mobile bed with an optimum yield, as the personskilled in the art knows how to do, the amount of extract injected hadin that case to be multiplied by a factor of correction of dischargeflow β, equal to 1.5.

A productivity of the less immobilised enantiomer equal to 10.3 mg/minwith a purity of 99.1% was obtained.

FIG. 5 shows the curves of variation in the concentration C of moreimmobilised enantiomer (curve a) and of less immobilised enantiomer(curve b) in simulated mobile bed devices respectively according to theprior state of the art (curves in broken lines) and according to theinvention (curves in solid lines), at the level of the inlet of each ofthe eight columns C1 to C8 of the device.

In order to increase the amount of charge with respect to the referencestate, one can play, as has been mentioned previously, on the flow ofthe charge by increasing said flow, but a charge flow identical to thatof the reference state may equally well be conserved and theconcentration of the charge increased.

All the experiments carried out by Applicants enabled them to ascertain,within the framework of the present invention, that the factor ofcorrection of discharge flow β depended on the selectivity, i.e. acoefficient representative of the respective affinity of the twoenantiomers for the stationary phase. It is known that the selectivity αis defined as follows: $\alpha = \frac{{t2} - {t0}}{{t1} - {t0}}$

where:

t₂ is the time of retention of the more immobilised enantiomer

t₁ is the time of retention of the less immobilised enantiomer

t₀ to is the nonproductive time of the chromatography column inquestion, i.e. the time of passage of the fluid in the dead volume ofthe column.

The curves of variation of the factor of correction of discharge flow β,as a function of the factor of increase of the amount of charge γ forvarious values of the selectivity α and in particular for the values ofthe enantiomers studied, were established and shown in FIG. 6, namely:

Curve Ref. Enantiomer Selectivity a Methyl 2-cyano-5-phenyl pentanoate1.1 b Racemic ester 1.3 c Binaphtol 1.4 d(+/−)-5-[1,2,3,4-tetrahydro-6-chinolyl]- 1.86-methyl-3,6-dihydro-2H-1,3,4-thiadiazin-1-on e Methsuximide 2.8

In a first approximation, in a relatively narrow range, for example forfactors of increase of the amount of charge γ included between 2 and 3,it may be considered that the coefficients β and γ are bound by asubstantially linear mathematical relationship. The slope of the curvesα=f(γ) (represented in FIG. 6) thus varies approximately between 0.38for a value of selectivity α of 1.1 to 0 for a value of selectivity α of2.8.

More precisely, by the numerous tests that they made, Applicantsestablished that the curve (FIG. 7) representing the variation of theratio of the factor of increase of the amount of charge γ with respectto the factor of correction of the discharge flow α decreased by a valueof one (viz the value γ/α−1) as a function of the factor of increase ofthe amount of charge γ minus one (viz the value γ−1) was a straightline. It was also established that the slope of this straight linedepended on the value of the selectivity a of the stationary phasehaving regard to the two enantiomers in question. More precisely, it wasestablished that this slope (as represented in FIG. 8) was substantiallyequal to a quarter of the selectivity α of the stationary phase (viz thevalue 0.25 α).

According to the invention, γ and β are thus substantially bound by theformula:

(γ/β−1)=0.25.α(γ−1)  (1)

The method according to the invention thus makes it possible to enrichtwo optical isomers constituted by a mixture of these isomers with amuch improved productivity. In the majority of industrial methods ofchiral separation, an optical isomer must be produced with a very highoptical purity. Two principal cases of application may thus be defined:

in a first variant embodiment, the optical isomer which it is desired toproduce is enriched to a purity less than that required. The step ofenrichment according to the invention may be followed by an additionalseparation step such as a crystallization or an enantioselectiveresolution by simulated mobile bed with a chiral or achiral stationaryphase, so that the overall treatment, namely the one according to theinvention, completed by a subsequent treatment provides a result betterthan that of a one-step method such as a simulated mobile bed with achiral stationary phase,

in a second variant, the non-desired optical isomer is enriched to apurity less than that finally required.

The present invention may be followed by an additional treatment step,such as a racemization, making it possible to recycle a mixture ofoptical isomers in the method according to the invention from theenriched and non-desired optical isomer.

The present invention may, of course, also be carried out when it isdesired to increase the productivity and purity of the more immobilisedenantiomer. The modus operandi would in that case be the same aspreviously, except that, in that case, the amount of extract would bedecreased by dividing it by the factor of correction of discharge flowβ.

Although the present invention has been described having regard toapplications developed with simulated mobile bed devices with fourzones, the person skilled in the art will, of course, be able to applythe teaching of the present invention to other models of simulatedmobile beds such as in particular devices with three zones.

What is claimed is:
 1. Method of separation of a mixture of two opticalisomers adapted to be more or less immobilised in a chiral stationaryphase, this method being of the type employing a simulated mobile bed,constituted by an assembly of columns (C1, C2, C3, C4, C5, C6, C7, C8)disposed in series, filled with a chiral stationary phase, which aredivided into at least three successive zones (I, II, III), namely afirst zone (I) at the inlet of which an eluent is injected and at theoutlet of which a flux, called discharge flux, principally containingthe more immobilised enantiomer, is drawn off, a second zone (II) at theoutlet of which said mixture is injected, and a third zone (III) at theoutlet of which a flux, called raffinate flux, principally containingthe less immobilized enantiomer is drawn off, characterized in that,with respect to a reference state in which: a) the optical isomer moreimmobilised in the stationary phase is completely desorbed in the firstzone (I) and is completely adsorbed in the third zone (III) and wherethe optical isomer less immobilised in the stationary phase iscompletely desorbed in the second zone (II), and b) the amount of chargeis maximum, the amount of charge injected is greater than that of thereference state, the discharge flow is greater than that of thereference state if it is desired at least to preserve the purity of theless immobilised enantiomer, or the discharge flow is less than that ofthe reference state if it is desired at least to preserve the purity ofthe more immobilised enantiomer.
 2. Method according to claim 1,characterized in that the amount of charge injected is rendered greaterthan that of the reference state by playing on the volume flow of thecharge.
 3. Method according to claim 1, characterized in that the amountof charge injected is rendered greater than that of the reference stateby playing on the concentration of the charge.
 4. Method according toclaim 1, characterized in that a simulated mobile bed comprising fourzones (I, II, III, IV) is used, the fourth zone (IV) being locateddownstream of the drawing-off point (13) of raffinate and, in thereference state, the less immobilised optical isomer in the stationaryphase is completely adsorbed in this fourth zone (IV).
 5. Methodaccording to claim 1, characterized in that the outlet of the last zoneis connected to the inlet of the first zone.
 6. Method according toclaim 1, characterized in that, at first approximation, the factor ofincrease of amount of charge (γ), i.e. the ratio of the amount of chargeinjected with respect to the amount of charge injected in the referencestate, for narrow intervals of this factor, and the factor of correctionof discharge flow (β), i.e. the ratio of the discharge flow with respectto the ratio of the discharge flow in the reference state, are bound bya substantially linear mathematical relationship.
 7. Method according toclaim 6, characterized in that the slope of the curve representing thevariation of the factor of correction of the discharge flow (β) as afunction of the variation of the factor of increase of amount of charge(γ) varies inversely with respect to the value of the selectivity (α) ofthe stationary phase used.
 8. Method according to claim 1, characterizedin that the factor of increase of the amount of charge (γ) and thefactor of correction of discharge flow (β) are bound by a linearrelationship expressing the ratio of the factor of increase of theamount of charge with respect to the factor of correction of dischargeflow (γ/β) decreased by a value of one (γ/β−1), as a function of thefactor of correction of charge flow (γ) decreased by a value of one. 9.Method according to claim 8, characterized in that the factor ofproportionality of said linear relationship comprises the value of theselectivity (α) of the stationary phase used.
 10. Method according toclaim 9, characterized in that said factor of proportionality issubstantially equal to a quarter of the value of the selectivity (α).11. Method according to one of claims 8 to 10, characterized in that thefactor of increase of the amount of charge (γ), the factor of correctionof discharge flow (β) and the selectivity (α) are substantially bound bythe relationship: (γ/β−1)=0.25.α(γ−1).